Field of the Invention
The present invention relates to an apparatus for collecting a peptide fragment, more specifically an apparatus for collecting the carboxyl terminal peptide fragment from a mixture of peptide fragments resulting from specific cleavage of the peptide bond between a lysine residue and the carboxyl terminal amino acid residue adjacent thereto in a polypeptide.
Discussion of the Related Art
Methods for analysis of the carboxyl terminal of a peptide (hereinafter referred to as "C-terminal") include the hydrazinolysis method, the tritium labeling method and the carboxypeptidase method. Both the hydrazinolysis method and the tritium labeling method are for determination of the end C-terminal amino acid residue only, none of which can determine all C-terminal amino acid residues. The carboxypeptidase method is based on the sequential peptide bond cleavage from the C-terminal of a polypeptide by various carboxypeptidases. Generally, this method has drawbacks such as troublesome operation, large sample volume requirements uncertainty in the amino acid sequencing and poor applicability to polypeptides having relatively long chains.
A method for peptide fragment collection for solving this problem is disclosed in Japanese Patent Laid-Open No. 1-235600. In this method, peptide bonds between lysine residues and C-terminal amino acid residues adjacent thereto are selectively cleaved. The resulting peptide fragment mixture is reacted with a solid support having on its surface a functional group capable of forming a covalent bond upon reaction with a free amino group. Subsequently, the peptide bond between the terminal residue having the amino group and the residue adjacent thereto is cleaved with an acid such as trifluoroacetic acid (TFA) and a 50%. acetonitrile-2-propanol mixture containing 0.1% TFA is added, followed by centrifugation.
The C-terminal peptide thus separated is collected and then dried in a vacuum. In this method, the C-terminal peptide is bound to the solid support at its .alpha.-amino group alone, while the other peptides are bound to the solid support at the .epsilon.-amino group of the lysine residue as well as at the N-terminal amino group. When this peptide-solid support complex is treated with an appropriate acid under appropriate conditions, the peptide bond between the amino-terminal residue and the adjacent residue alone is cleaved, and the C-terminal peptide is separated in the reaction solution with its amino-terminal residue left on the solid support, while the other peptides remaining bound to the solid support (FIG. 17).
Although the above-described method permits relatively easy separation and collection of the C-terminal peptide, it still requires skillful manual operation in several processes, including centrifugation and vacuum-drying, to collect the C-terminal peptide fragment from the peptide fragment mixture obtained by the enzyme treatment. This makes it difficult to collect the C-terminal peptide with simple operation and high accuracy, and reproducibility is low.